There has long been an interest in the development of assay systems which can determine the presence or amount of specific substances in samples derived from biological specimens. Over the past two decades, immunoassays, which employ naturally occurring receptors directed to specific target substances, have provided valuable diagnostic tools for detecting substances of clinical significance. There are numerous immunoassays in the prior art in which one component of an immunological pair, e.g., an antigen or antibody, is detected or measured by using the complementary partner labelled with a tag which provides a detectible signal.
In one assay technique, known as competitive binding technique, the substance to be detected competes with a labelled reagent of the same substance for a limited number of receptor sites. For example, for the detection of an unknown amount of a selected antigen in a liquid sample, a known amount of the labelled antigen is added to the sample and then contacted with receptor antibody specific for the antigen. The amount of labelled antigen which binds to the antibody is inversely proportional to the amount of the unknown antigen in the sample.
In another assay, known as a sandwich assay, receptor antibody is bound to a solid surface and the selected antigen in the sample binds to that antibody. A second labelled antibody capable of binding to the bound antigen is then reacted with the antigen to form an immobilized reaction product. The label in the reaction product is detected as an indication of the presence of the antigen in the sample.
For the detection or measurement of an antigen using a sandwich technique, antisera have been used for many years for both the labelled antibody and for the receptor antibody on the surface. More recently, monoclonal antibodies have been used in place of the antisera in such assay. In one such system, described in Wada, et al., Clin. Chem., 28(9):1986-1966 (1982), the receptor antibody was directed to one subunit of a particular antigen, hCG, while an enzyme-labelled monoclonal antibody was directed to another subunit. In this assay, the receptor antibody is immobilized on the inside of the test tube to which the sample was added.
Reaction on a solid surface can be relatively slow because the contact between the immobilized reagent and the analyte in the sample is limited. The assay time has been reduced by immobilizing the receptor antibody within a porous membrane, exposing the antibody molecules in a three-dimensional matrix. In many such systems the liquid sample containing the target antigen is drawn through the membrane into an underlying absorbent material. One such system, disclosed for use in a competitive binding assay, is U.S. Pat. No. 3,888,629. Other systems disclosed for use in competitive or sandwich assays include U.S. Pat. Nos. 4,246,339, 4,366,241, 4,632,901.
However, most such assay systems allow the deposition of particulate matter on the surface of the filter, thus obscuring the results when using, for example, a chromophoric label. In addition, these systems do not provide a means for controlling the flow rate of the liquid in the sample. The system of U.S. Pat. No. 4,623,461 attempts to address these problems by forcing the liquid sample to flow transversely to the periphery of the filter by having the absorbent material associated only with the peripheral area of the filter. It is claimed that this system results in decreased deposition of particulate matter on the filter surface and improved separation of the analyte.
Unfortunately, as the frontal surface area of the transverse cross-section of the membrane is quite small, the liquid will necessarily take longer to pass completely through into the absorbent. In addition, the flow through characteristics could not be altered without changing to a different membrane.
Thus, it would be desirable to improve the sensitivity and separation of a membrane-bound receptor assay while controlling the fluid flow characteristics of the assay system.
It would also be desirable to control the fluid flow characteristics of a system by means other than the thickness and pore size of the membrane which immobilizes the reactant.